Success of organ and tissue transplantation is limited by side effects of long-term immunosuppressive therapy and late graft rejection, which occurs despite global immunosuppression. Allograft tolerance would obviate the need for long-term immunosuppressive therapy while preventing rejection. Proof of principle that mixed chimerism can lead to organ allograft tolerance across MHC barriers in non-human primates (NHPs) and patients has been obtained using combined kidney and bone marrow transplantation (CKBMT). However, mixed chimerism was only transient and tolerance was not been achieved to other organs such as islet, liver, heart or lung allografts with this transient chimerism approach in NHPs. Studies in mice indicate that durable mixed chimerism is associated with robust, systemic tolerance that permits survival of the most challenging allografts. Thus, reliable methods of achieving durable mixed chimerism without GVHD, the major complication of hematopoietic cell transplantation (HCT) in humans, are needed to advance the use of mixed chimerism to achieve tolerance for all types of organ and tissue allografts. We have obtained preliminary evidence that the addition of expanded recipient regulatory T cells (Tregs) to donor bone marrow infusion results in more durable mixed chimerism and more robust tolerance than is induced by BMT alone in NHPs receiving a low-toxicity, non-myeloablative BMT regimen that has been completely free of GVHD. We now aim to: 1) Develop an optimal regimen combining non-myeloablative conditioning, infusion of expanded recipient Tregs and transient post-transplant immunosuppression for the induction of durable mixed allogeneic chimerism in cynomolgus monkeys. We will improve upon the prolonged, high level chimerism observed with the addition of expanded recipient Tregs to a protocol using 28 days of post-BMT rapamycin. We will attempt to enhance donor hematopoietic stem cell (HSC) survival by delaying BM infusions to minimize deleterious effects of ATG on stem cells and by enhancing initial immunosuppression. We will then identify the minimal Treg dose required to achieve durable chimerism. Donor skin grafted 4 months post- BMT will be used as a robust test of tolerance; 2) Assess the persistence of Tregs and mechanisms of tolerance in durable mixed chimeras prepared in AIM 1. The roles of regulatory mechanisms and deletion in maintaining tolerance will be assessed. The ability to safely and reliably achieve durable mixed chimerism across MHC barriers with non-myeloablative conditioning, without GVHD, in a NHP model will be a major breakthrough. Success of these studies will allow the achievement of tolerance to all types of organ and tissue allografts in humans and will increase the safety of HCT for the treatment of non-malignant inherited hematological disorders and autoimmune diseases that are correctable by a state of durable mixed chimerism.